1 PROJECT SUMMARY 2 Defective-RNAs (D-RNAs) are versions of RNA viral genomes that arise naturally during viral infections but have 3 been truncated or rearranged by non-homologous recombination. While not encoding for functional viruses, they 4 can be amplified and co-passaged with the wild-type virus. D-RNAs have been observed and characterized for 5 multiple decades in multiple viral systems. Their preponderance and the conserved nature of their genetic 6 structures suggests that their emergence during viral infections is not spontaneous or stochastic, but may affect 7 the fitness of a viral species. Recently, the roles of D-RNAs in modulating the outcome of a viral infection in 8 patients has been demonstrated, both in ?wild? infections and during vaccination. 9 Using a combination of short-read (ClickSeq) and long-read (Oxford Nanopore) next-generation sequencing, we 10 have previously characterized the emergence and accumulation of specific sets of D-RNAs in the prototypical 11 nodavirus, Flock House virus (FHV) (Jaworski et al. PLoS Path 2017). In subsequent studies, we have 12 characterized the host transcriptional response to FHV both in the presence and absence of D-RNAs. We have 13 recently developed novel and powerful pipelines to perform differential gene expression and alternative 14 polyadenylation using Poly(A)-ClickSeq (Routh., G3 2019; Elrod et al. Methods 2019). We have also developed 15 computational pipelines to characterize and single-cell RNAseq (scRNAseq) data that integrates our bespoke 16 Virus Recombination Mapping (ViReMa) algorithm to report the abundance of D-RNAs within single cells for 17 clustering analysis. Using these novel approaches, we found that infection with FHV results in an upregulation 18 of the unfolded protein response including heat-shock factors. Surprisingly, when D-RNAs were present in the 19 viral inoculum, rather than the expected suppression of the host response, expression of heat-shock factors was 20 strongly enhanced. We also observed that non-coding RNAs including snRNA:7SK and hsromega were 21 upregulated. These are abundant and well-characterized ncRNAs with roles in the regulation of stress-response 22 genes. Furthermore, heat-shock factors have previously been demonstrated to promote FHV replication and to 23 be pro-viral host factors. We therefore posit that the emergence of D-RNAs in FHV is a pro-viral phenomenon 24 (despite the reduction in specific infectivity of the viral inoculum) through stimulation of the heat-shock response 25 and UPR. 26 We hypothesize that the emergence of D-RNAs in FHV is under positive selection and these species constitute 27 ?Defective-enhancing RNAs?. Characterizing the mechanism of the activity of these Defective-enhancing RNAs 28 would constitute a novel paradigm and provide a framework for future studies elucidating the roles of Defective- 29 RNAs in a broad range of RNA viruses. By characterizing how they modulate the host cell immune responses 30 we will therefore understand the selective forces that drive the emergence and amplification of Defective-RNAs.